Regulation of cholinesterase gene expression affects neuronal differentiation as revealed by transfection studies on reaggregating embryonic chicken retinal cells.
In the embryonic chicken neuroepithelium, butyrylcholinesterase (BChE) as a proliferation marker and then acetylcholinesterase (AChE) as a differentiation marker are expressed in a mutually exclusive manner. These and other data indicate a coregulation of cholinesterase expression, and also possible roles of cholinesterases during neurogenesis. Here, both aspects are investigated by two independent transfection protocols of dissociated retina cells of the 6-day-old chick embryo in reaggregation culture, both protocols leading to efficient overexpression of AChE protein. The effect of the overexpressed AChE protein on the re-establishment of retina-like three-dimensional networks (so-called retinospheroids) was studied. In a first approach, we transfected retinospheroids with a pSVK3 expression vector into which a cDNA construct encoding the entire rabbit AChE gene had been inserted in sense orientation. As detected at the mRNA level, rabbit AChE was heterologously overexpressed in chicken retinospheroids. Remarkably, this was accompanied by a strong increase in endogenous chicken AChE protein, while the total AChE activity was only slightly increased. This increase was due to chicken enzyme, as shown by species-specific inhibition studies using fasciculin. Clearly, total AChE activity is regulated post-translationally. As an alternative method of AChE overexpression, transfection of spheroids was performed with an antisense-5'-BChE vector, which not only resulted in the down-regulation of BChE expression, but also strongly increased chicken AChE transcripts, protein and enzyme activity. Histologically, a higher concentration of AChE protein (as a consequence of either AChE overexpression or BChE suppression) was associated with an advanced degree of tissue differentiation, as detected by immunostaining for the cytoskeletal protein vimentin.
Robitzki
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Department of Developmental and Neurobiology, Institute for Zoology, University of Technology, Darmstadt, Germany.
Mack
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Hoppe
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Chatonnet
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D000818:Animals; D002449:Cell Aggregation / Q000502:physiology; D002454:Cell Differentiation; D002478:Cells, Cultured; D002642:Chick Embryo; D002802:Cholinesterases / Q000096:biosynthesis* / Q000235:genetics*; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000096:biosynthesis; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007150:Immunohistochemistry; D009474:Neurons / Q000201:enzymology / Q000502:physiology*; D016376:Oligonucleotides, Antisense; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D011817:Rabbits; D012160:Retina / Q000166:cytology / Q000196:embryology / Q000201:enzymology*; D014162:Transfection
D005136:Eye Proteins; D016376:Oligonucleotides, Antisense; D012313:RNA; D004247:DNA; D002802:Cholinesterases